Exon skipping is a common result of splice mutations and has been reported in a wide variety of genetic disorders1, yet the underlying mechanism is poorly understood. Often, such mutations are incompletely penetrant, and low levels of normal transcript and protein are maintained1 Familial dysautonomia (FD) (MIM#2239001), also known as Riley Day syndrome or hereditary sensory and autonomic neuropathy III (HSAN-III), is the best-known and most common member of a group of congenital sensory and autonomic neuropathies (HSAN) characterized by widespread sensory and variable autonomic dysfunction (Axelrod F B: (1996) Autonomic and Sensory Disorders. In: Principles and Practice of Medical Genetics, 3rd edition, AEH Emory and DL Rimoin eds. Churchill Livingstone, Edinburgh. pp 397-411; Axelrod F B (2002) Hereditary Sensory and Autonomic Neuropathies: Familial Dysautonomia and other HSANs. Clin Auton Res 12 Supplement 1, 2-14). FD affects neuronal development and is associated with progressive neuronal degeneration. Multiple systems are impacted resulting in a markedly reduced quality of life and premature death (Axelrod F B: (1996) Autonomic and Sensory Disorders. In: Principles and Practice of Medical Genetics, 3rd edition, AEH Emory and DL Rimoin eds. Churchill Livingstone, Edinburgh. pp 397-411; Axelrod F B (2002) Hereditary Sensory and Autonomic Neuropathies: Familial Dysautonomia and other HSANs. Clin Auton Res 12 Supplement 1, 2-14).
FD is a recessive disorder that has a remarkably high carrier frequency of 1 in 30 in the Ashkenazi Jewish population5. FD is caused by mutations in the IKBKAP gene2,3 (Genbank Accession No. NM—003640.), and all cases described to date involve an intron 20 mutation that results in a unique pattern of tissue-specific exon skipping. Accurate splicing of the mutant IKBKAP allele is particularly inefficient in the nervous system. Three FD mutations have been identified in the I-k-B kinase (IKK) complex-associated protein (IKBKAP): IVS20+6T→C, which leads to variable, tissue-specific skipping of exon 20 (FIG. 1a), R696P, and P914L2,3,6. All FD patients tested to date carry at least one IVS20+6T→C mutation, with more than 99.5% being homozygous, and the remainder being heterozygous with either R696P or P914L on the alternate allele.
The IVS20+6T→C mutation does not cause complete loss of function. Instead, it results in a tissue-specific decrease in splicing efficiency of the IKBKAP transcript; cells from patients retain some capacity to produce normal mRNA and IKAP protein (Slaugenhaupt et al. (2001) Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. Am J Hum Genetics 68:598-605). The mRNA is widely distributed. Highest levels are in the nervous system, but substantial amounts are also present in peripheral organs (Mezey et al. (2003) Of splice and men: what does the distribution of IKAP mRNA in the rat tell us about the pathogenesis of familial dysautonomia? Brain Research 983:209). It has been reported previously that all FD tissues tested express both wild-type (WT) and mutant (MU) IKBKAP mRNA2,4. The effect of the most common (splicing) mutation varies from tissue to tissue—neuronal tissues seem primarily to express mutant mRNA; somatic tissues express roughly equal levels of normal and mutant mRNA. Accurate measurement of the ratio of the two mRNA species using both densitometry and real-time quantitative PCR has revealed that the levels of WT IKBKAP mRNA vary between tissues and are lowest in central and peripheral nervous systems4. This leads to a drastic reduction in the amount of IKAP protein in these tissues.
There are other disorders that are caused, at least in part, by missplicing including Neurofibromatosis 1 (NF1), also known as von Recklinghausen NF or Peripheral NF. NF1 occurs in 1:4,000 births and is characterized by multiple cafe-au-lait spots and neurofibromas on or under the skin. Enlargement and deformation of bones and curvature of the spine may also occur (Riccardi, 1992, Neurofibromatosis: phenotype, natural history, and pathogenesis. 2nd ed. Baltimore: Johns Hopkins University Press). Occasionally, tumors may develop in the brain, on cranial nerves, or on the spinal cord. About 50% of people with NF also have learning disabilities (Chapter 6 in Rubenstein and Korf, 1990, Neurofibromatosis: a handbook for patients, families, and health-care professionals. New York: Thieme Medical Publishers).
The NF1 gene was identified and the protein product characterized in 1990 (Cawthon et al., 1990, Cell 62: 193-201; Wallace et al., 1990, Science 249:181-6). The entire sequence of the expressed NF1 gene has been reported (Viskochil et al., 1993, Annu Rev Neurosci 16: 183-205; Gutmann and Collins, 1993, Neuron 10: 335-43; Genbank Accession No. NM—000267). The gene is has at least 59 exons and codes for a 2818 amino acid protein called neurofibromin. To date, 180 different NF1 mutations have been identified. The NF1 Genetic Analysis Consortium maintains a database of mutations identified in more than 45 collaborating laboratories throughout the world. According to data from the Consortium, the NF1 mutations described to date include 4 chromosomal rearrangements, 89 deletions (14 deletions involving the entire gene, 35 deletions involving multiple exons, and 37 small deletions), 23 insertions (3 large and 20 small), 45 point mutations (29 stop mutations and 16 amino acid substitutions), and 18 intronic mutations affecting splicing, and 4 mutations in 3′ untranslated region of the gene. About 30% of NF1 patients carry a splice mutation resulting in the production of one or several shortened transcripts (Vandenbroucke et al., 2002, BMC Genomics 3:13 and Serra et al., 2001, Hum Genet. 108:416-29).
Cytokinins are a class of plant hormones defined by their ability to promote cell division in plant tissue explants in the presence of an auxin, such as indoleacetic acid, and nutrients, including vitamins, mineral salts, and sugar. In promoting cell division of plant cells, cytokinins are active at low concentrations (as low 0.01 parts per million (ppm)), but exhibit activity only in the presence of an auxin. Certain cytokinins, including zeatin and 6-(3,3-dimethylallyl)-aminopurine, also occur as the base moiety components of transfer RNA in yeast, bacterial, animal cells and plant cells. The cytokinin kinetin (6-furfuryl-aminopurine) forms complexes with certain RNA-binding proteins of wheat embryo extracts and appears to promote protein synthesis in plants (see, e.g., Spirin and Ajtkhozhin (1985) Trends in Biochem. Sci., p. 162). Kinetin and other cytokinins are used in conjunction with auxin used in horticulture and in plant tissue culture, such as in the production of plantlets from plant callus tissue. Cytokinins are also used in the production of protein-rich yeast (see e.g., East German Patent No. 148,889 (1981) (Derwent World Patent Index Abstract)) and to augment the growth of microbial cultures (Merck Index, 10th Ed. (1983) Entry 5148, Merck and Co., Rahway, N.J., U.S.A.).
Kinetin belongs to the family of N6-substituted adenine derivatives known as cytokinins, or plant growth factors, that also includes zeatin, benzyladenine and 2iP (FIG. 1b). Kinetin is also known as 6-furfurylaminpurine (C10H9N5) and has a molecular weight of 215.21 (Soriano-Garcia and Parthsarathy, 1975, Biochem Biophys Res Commun 64:1062-8). Kinetin is currently marketed as an anti-aging ingredient in skin treatments due to its ability to ameliorate aging characteristics in cultured human fibroblasts8, possibly through anti-oxidant activity9.
Certain cytokinins have been shown to inhibit the growth of tumor cells in vitro (see, e.g., Katsaros et al. (1987) FEBS Lttrs. 223:97-103). It appears that this effect is mediated via the cytotoxic affects of adenosine analogs, such as the 6-(substituted amino) purine cytokinins, that interfere with tRNA methylating enzymes (Wainfan et al, (1973) Biochem. Pharmacol. 22:493-500). When immortalized fibroblast cells are contacted with adenosine analogs the cultured cells exhibit decreased growth rate and a change in morphology from the normal flattened elongated morphology typical of cultured fibroblasts to a very elongated spindle-shape characteristic of a cytotoxic response. The very elongated shape of immortalized cells exhibiting this response is not shape characteristic of young, healthy, primary cultures of normal diploid fibroblasts.
Kinetin has been shown to be capable of delaying or preventing a host of age-related changes of human skin fibroblasts grown in laboratory culture which has led to its incorporation into topical skin products. West MID (1994) The cellular and molecular biology of skin aging. Arch Dermatol 130:87-95. Fibroblasts, which produce collagen and elastin, have been shown to decrease in number and vitality as skin ages not only in vitro, but also in vivo. The number of fibroblasts decreases at least 50% between birth and the age of 80 years. West MID (1994) The cellular and molecular biology of skin aging. Arch Dermatol 130:87-95. Rattan S I, Clark B F. (1994) Kinetin delays the onset of aging characteristics in human fibroblasts. Biochem Biophys Res Commun 201:665-72. Kinetin has been shown to delay or prevent a range of cellular changes associated with in vitro aging of human skin cells, including alterations in cell morphology, growth rate, size, cytoskeletal organization, macromolecular synthetic activity and accumulation of lipofuscin aging pigments but kinetin did not alter the maximum in vitro life span of human skin cells or their ability to multiply in culture. Rattan S I, Clark B F. (1994) Kinetin delays the onset of aging characteristics in human fibroblasts. Biochem Biophys Res Commun 201:665-72. Thus, kinetin was devoid of activities associated with cellular immortalization, malignant transformation and carcinogenesis. Rattan S I, Clark B F. (1994) Kinetin delays the onset of aging characteristics in human fibroblasts. Biochem Biophys Res Commun 201:665-72.
Using rats as a mammalian model, it has been shown that plant cytokinins can affect lipid peroxidation in erythrocyte, muscle, liver, heart and kidney tissue, Celik I, Tuluce Y, Ozok N. (2002) Effects of indoleacetic acid and kinetin on lipid peroxidation levels in various rat tissues. Turk J Biol 26; 193-196. Celik et al. (2002) showed that kinetin had much less toxicity as compared to indoleacetic acid (IAA), when administered orally to rats. Celik I, Tuluce Y, Ozok N. (2002) Effects of indoleacetic acid and kinetin on lipid peroxidation levels in various rat tissues. Turk J Biol 26; 193-196. It was shown that IAA interacts primarily with the liver and kidney tissue cells, resulting in lipid peroxidation synthesis, whereas kinetin had no such effect in the liver or kidney.